RESUMEN
The nongenotropic ligand estren (Science 298:843-846, 2002) was evaluated for its transcriptional activity mediated by the human androgen receptor (AR). Our results show that estren can bind, translocate, transactivate, and regulate two known target genes of AR in androgen-responsive cell lines. Estren binds recombinant AR with 10-fold higher affinity than either estrogen receptor (ER)-alpha or ERbeta. Estren-bound AR can translocate AR to the nucleus and stimulate the androgen response element-luciferase reporter activity with an efficacy similar to that of androgen. Estren also increased the expression of prostate-specific antigen (PSA) in a dose-dependent manner in human LnCaP cells. Using chromatin immunoprecipitation analysis, we show that the estren-bound AR coimmunoprecipitates with a region of the PSA gene promoter. Therefore, cotreatment with an AR antagonist, bicalutamide, blocked the estren-induced increase in PSA expression. In contrast, phosphoinositol 3-kinase inhibitor wortmannin, or extracellular signal-regulated kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene (U0126), and ER antagonist ICI-182780 failed to block the effects of estren. In vivo analysis of estren's action on male-orchidectomized ICR mice revealed estren's AR agonist actions on the levator ani and seminal vesicle target tissues. Taken together, our results reveal the hitherto unidentified genotropic action of estren mediated by AR in androgen-responsive cells and tissues.
Asunto(s)
Estrenos/metabolismo , Estrenos/farmacología , Receptores Androgénicos/metabolismo , Andrógenos , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Cinética , Masculino , Antígeno Prostático Específico/análisis , Neoplasias de la Próstata , Transporte de Proteínas , ARN Mensajero/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Propionic acid derivative 8, which was designed and synthesized based on putative pharmacophores of known PPARgamma- and PPARalpha-selective compounds, exhibits potent dual PPARalpha/gamma agonist activity as demonstrated by in vitro binding and dose overlap in the newly introduced EOB mouse model for glucose lowering and lipid/cholesterol homeostasis.
Asunto(s)
Hipoglucemiantes/síntesis química , Hipoglucemiantes/farmacología , Propionatos/síntesis química , Propionatos/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Factores de Transcripción/agonistas , Animales , Glucemia/metabolismo , HDL-Colesterol/sangre , Diabetes Mellitus/sangre , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus/genética , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diseño de Fármacos , Ratones , Ratones Endogámicos , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Triglicéridos/sangreRESUMEN
A 400 bp PCR product generated with degenerate primers derived from the glucagon-like peptide-1 receptor was used to screen a rat skeletal muscle cDNA library. The predicted amino acid sequence of the 978 bp open reading frame has a predicted M(r) of 35 804, an estimated isoelectric point (pI) of 5.31 and contains seven WD-40 repeats, which are common to G-protein beta subunits (Gbeta). Although chemically and structurally similar to Gbeta subunits, the predicted amino acid sequence, when compared with the previously cloned Gbeta isoforms, was found to be only 31-41% similar and thus was named Gbeta-like (GbetaL, 'Gable'). Western blotting of whole-cell lysates and immunoprecipitates of membrane and cytosolic fractions of HEK 293 cells stably overexpressing a carboxy-terminal His-tagged GbetaL indicates that the protein is cytosolic and that it migrates at 42 kDa. A 4 kb transcript was detected in all tissues surveyed by northern blotting; however, an additional 2 kb transcript was detected in testis. Expression of GbetaL mRNA was highest in the brain and testis, followed by lung, heart, kidney, skeletal muscle, spleen and liver. In addition, reverse transcriptase/PCR showed that several other tissues and cell lines express GbetaL. The ubiquitous nature of the tissue expression pattern of GbetaL is similar to that of the insulin receptor, which suggests that insulin may influence GbetaL expression. Indeed, GbetaL protein and mRNA levels, in fully differentiated 3T3-L1 adipocytes, were upregulated by insulin in a concentration-dependent fashion. These changes were highly sensitive to insulin stimulation, being minimally affected by doses as low as 0.1 nM and maximally elevated by 1 nM doses. These data suggest that insulin regulates GbetaL production and imply that some of the actions of insulin may be mediated, in part, by this novel intracellular protein.
Asunto(s)
Adipocitos/metabolismo , Insulina/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , ADN Complementario/genética , Relación Dosis-Respuesta a Droga , Expresión Génica , Biblioteca de Genes , Proteínas de Unión al GTP Heterotriméricas/biosíntesis , Proteínas de Unión al GTP Heterotriméricas/genética , Insulina/farmacología , Datos de Secuencia Molecular , ARN Mensajero/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución Tisular , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The cytoplasmic domain of the insulin receptor (IR) beta-subunit contains cysteine (Cys) residues whose reactivity and function remain uncertain. In this study, we examined the ability of the bifunctional cross-linking reagent 1,6-bismaleimidohexane (BMH) to covalently link IR with interacting proteins that possess reactive thiols. Transfected Chinese hamster ovary cells expressing either the wild-type human IR, C-terminally truncated receptors, or mutant receptors with Cys --> Ala substitutions and mouse 3T3-L1 adipocytes were used to compare the BMH effect. The results showed the formation of a large complex between the wild-type human receptor beta-subunit and molecule X, a thiol-reactive membrane-associated protein, in both intact and semipermeabilized cells in response to BMH. Prior cell stimulation with insulin had only a modest effect in this process. Western blot analysis revealed that the receptor alpha-subunit was not present in the beta-X complex. The BMH cross-linking did not inhibit in vitro tyrosine phosphorylation of the receptor complexed with molecule X. Both the human IR Cys981Ala mutant and murine IR, that lacks the equivalent of human Cys(981), failed to react with BMH. Finally, no covalent association between IR beta-subunit and IRS-1, the protein tyrosine phosphatase LAR or SHP-2 was observed in BMH-treated cells expressing the wild-type human IR. These results demonstrate a striking difference in reactivity among the cytoplasmic IR beta-subunit thiols and clearly show that Cys(981) of human IR beta-subunit is in close proximity to a thiol-reactive membrane-associated protein under basal and insulin-stimulated conditions.
Asunto(s)
Cisteína/metabolismo , Maleimidas/metabolismo , Receptor de Insulina/metabolismo , Receptores de Superficie Celular , Células 3T3 , Animales , Células CHO , Cricetinae , Cisteína/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/metabolismo , Ratones , Mutación , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/metabolismo , Receptor de Insulina/genética , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores , Compuestos de Sulfhidrilo/metabolismo , TransfecciónRESUMEN
In the present study, we attempted to determine the importance of a 23-amino-acid sequence within the carboxyl terminus of the human insulin receptor (IR) molecule in modulating insulin action in Chinese hamster ovary cells. Stable expression of a minigene encoding the receptor fragment led to an increase in insulin-induced IR autophosphorylation that was 2.4-fold higher when compared to that of IR-expressing cells transfected with empty vector. Insulin-stimulated downstream signaling was also significantly elevated in cells expressing the minigene. It was found that expression of the minigene had no effect toward insulin-like growth factor I receptor kinase activity and function. These results indicate that the IR carboxyl terminus contains a motif that acts as a physiologic modulator of insulin signaling. J. Cell. Biochem. 78:160-169, 2000. Published 2000 Wiley-Liss, Inc.
Asunto(s)
Insulina/fisiología , Receptor de Insulina/química , Receptor de Insulina/fisiología , Animales , Células CHO , Cricetinae , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Cinética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fragmentos de Péptidos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Receptor de Insulina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , TransfecciónRESUMEN
In this study, AR42J pancreatic acinar cells were used to investigate if glucagon-like peptide-1 (GLP-1) or glucagon might influence amylase release and acinar cell function. We first confirmed the presence of GLP-1 receptors on AR42J cells by reverse trasncriptase-polymerase chain reaction (RT-PCR), Western blotting, and partial sequencing analysis. While cholecystokinin (CCK) increased amylase release from AR42J cells, GLP-1, alone or in the presence of CCK, had no effect on amylase release but both CCK and GLP-1 increased intracellular calcium. Similar to GLP-1, glucagon increased both cyclic adenosine monophosphate (cAMP) and intracellular calcium in AR42J cells but it actually decreased CCK-mediated amylase release (n = 20, P < 0.01). CCK stimulation resulted in an increase in tyrosine phosphorylation of several cellular proteins, unlike GLP-1 treatment, where no such increased phosphorylation was seen. Instead, GLP-1 decreased such protein phosphorylations. Genestein blocked CCK-induced phosphorylation events and amylase secretion while vanadate increased amylase secretion. These results provide evidence that tyrosine phosphorylation is necessary for amylase release and that signaling through GLP-1 receptors does not mediate amylase release in AR42J cells. J. Cell. Physiol. 181:470-478, 1999. Published 1999 Wiley-Liss, Inc.
Asunto(s)
Amilasas/metabolismo , Glucagón/farmacología , Fragmentos de Péptidos/farmacología , Precursores de Proteínas/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Secuencia de Bases , Señalización del Calcio/efectos de los fármacos , Línea Celular , Colecistoquinina/farmacología , AMP Cíclico/metabolismo , Cartilla de ADN/genética , Glucagón/fisiología , Péptido 1 Similar al Glucagón , Receptor del Péptido 1 Similar al Glucagón , Páncreas/citología , Páncreas/efectos de los fármacos , Páncreas/fisiología , Fragmentos de Péptidos/fisiología , Fosforilación , Precursores de Proteínas/fisiología , Ratas , Receptores de Glucagón/genética , Receptores de Glucagón/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tirosina/metabolismoRESUMEN
To identify novel seven transmembrane domain proteins from 3T3-L1 adipocytes, we used PCR to amplify 3T3-L1 adipocyte complementary DNA (cDNA) with primers homologous to the N- and C-termini of pancreatic glucagon-like peptide-1 (GLP-1) receptor. We screened a cDNA library prepared from fully differentiated 3T3-L1 adipocytes using a 500-bp cDNA PCR product probe. Herein describes the isolation and characterization of a 1.6-kb cDNA clone that encodes a novel 298-amino acid protein that we termed TPRA40 (transmembrane domain protein of 40 kDa regulated in adipocytes). TPRA40 has seven putative transmembrane domains and shows little homology with the known GLP-1 receptor or with other G protein-coupled receptors. The levels of TPRA40 mRNA and protein were higher in 3T3-L1 adipocytes than in 3T3-L1 fibroblasts. TPRA40 is present in a number of mouse and human tissues. Interestingly, TPRA40 mRNA levels were significantly increased by 2- to 3-fold in epididymal fat of 24-month-old mice vs. young controls as well as in db/db and ob/ob mice vs. nondiabetic control littermates. No difference in TPRA40 mRNA levels was observed in brain, heart, skeletal muscle, liver, or kidney. Furthermore, no difference in TPRA40 expression was detected in brown fat of ob/ob mice when compared with age-matched controls. Taken together, these data suggest that TPRA40 represents a novel membrane-associated protein whose expression in white adipose tissue is altered with aging and type 2 diabetes.
Asunto(s)
Adipocitos/química , Envejecimiento/metabolismo , Diabetes Mellitus/metabolismo , Epidídimo/química , Proteínas de la Membrana/análisis , Receptores de Glucagón/análisis , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/aislamiento & purificación , Receptor del Péptido 1 Similar al Glucagón , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Obesos/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas/análisis , Receptores de Glucagón/genéticaRESUMEN
Chinese hamster ovary (CHO) cells stably expressing the human insulin receptor and the rat glucagon-like peptide-1 (GLP-1) receptor (CHO/GLPR) were used to study the functional coupling of the GLP-1 receptor with G proteins and to examine the regulation of the mitogen-activated protein (MAP) kinase signaling pathway by GLP-1. We showed that ligand activation of GLP-1 receptor led to increased incorporation of GTP-azidoanilide into Gs alpha, Gq/11 alpha, and Gi1,2 alpha, but not Gi3 alpha. GLP-1 increased p38 MAP kinase activity 2.5- and 2.0-fold over the basal level in both CHO/GLPR cells and rat insulinoma cells (RIN 1046-38), respectively. Moreover, GLP-1 induced phosphorylation of the immediate upstream kinases of p38, MKK3/MKK6, in CHO/GLPR and RIN 1046-38 cells. Ligand-stimulated GLP-1 receptor produced 1.45- and 2.7-fold increases in tyrosine phosphorylation of 42-kDa extracellular signal-regulated kinase (ERK) in CHO/GLPR and RIN 1046-38 cells, respectively. In CHO/GLPR cells, these effects of GLP-1 on the ERK and p38 MAP kinase pathways were inhibited by pretreatment with cholera toxin (CTX), but not with pertussis toxin. The combination of insulin and GLP-1 resulted in an additive response (1.6-fold over insulin alone) that was attenuated by CTX. In contrast, the ability of insulin alone to activate these pathways was insensitive to either toxin. Our study indicates a direct coupling between the GLP-1 receptor and several G proteins, and that CTX-sensitive proteins are required for GLP-1-mediated activation of MAP kinases.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Páncreas/metabolismo , Receptores de Glucagón/metabolismo , Animales , Células CHO , Clonación Molecular , Cricetinae , Activación Enzimática , Receptor del Péptido 1 Similar al Glucagón , Humanos , Ratas , Receptor de Insulina/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The incretin hormone glucagon-like peptide-1 (GLP-1)-(7-36) amide is best known for its antidiabetogenic actions mediated via a GLP-1 receptor present on pancreatic endocrine cells. To investigate the molecular mechanisms of GLP-1 action in muscle, we used cultured L6 myotubes. In L6 myotubes, GLP-1 enhanced insulin-stimulated glycogen synthesis by 140% while stimulating CO2 production and lactate formation by 150%. In the presence of IBMX, GLP-1 diminished cAMP levels to 83% of IBMX alone. In L6 myotubes transfected with pancreatic GLP-1 receptor, GLP-1 increased cAMP levels and inhibited glycogen synthesis by 60%. An antagonist of pancreatic GLP-1 receptor, exendin-4-(9-39), inhibited GLP-1-mediated glycogen synthesis in GLP-1 receptor-transfected L6 myotubes. However, in parental L6 myotubes, exendin-4-(9-39) and GLP-1-(1-36) amide, an inactive peptide on pancreatic GLP-1 receptor, displaced 125I-labeled GLP-1 binding and stimulated glycogen synthesis by 186 and 130%, respectively. These results suggest that the insulinomimetic effects of GLP-1 in L6 cells are likely to be mediated by a receptor that is different from the GLP-1 receptor found in the pancreas.
Asunto(s)
Glucagón/farmacología , Glucógeno/biosíntesis , Músculo Esquelético/fisiología , Páncreas/fisiología , Fragmentos de Péptidos/farmacología , Precursores de Proteínas/farmacología , Receptores de Glucagón/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Animales , Carbacol/farmacología , Línea Celular , Clonación Molecular , AMP Cíclico/metabolismo , Glucagón/metabolismo , Glucagón/fisiología , Péptido 1 Similar al Glucagón , Receptor del Péptido 1 Similar al Glucagón , Glucólisis/efectos de los fármacos , Insulina/farmacología , Cinética , Músculo Esquelético/citología , Páncreas/metabolismo , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/fisiología , Precursores de Proteínas/metabolismo , Precursores de Proteínas/fisiología , Ratas , Receptores de Glucagón/biosíntesis , Proteínas Recombinantes/biosíntesis , Transfección , Ponzoñas/farmacologíaRESUMEN
We have identified in rabbit renal cells two alternatively spliced transcripts of the alpha-subunit rbslo1 and rbslo2. Rbslo1 has a novel "in-frame" 174-bp insertion immediately after the predicted S8 transmembrane segment, whereas rbslo2 has a 104-bp deletion between S9 and S10, creating a frameshift and a premature termination codon. Amino acid identity between mouse maxi K- channel alpha-subunit (mslo) and rbslol was 99%. Two transcript sizes of 4.2 and 7.5 kb were detected in brain, kidney, stomach, testis, and lung. Rbslo is expressed in glomeruli, thin limbs of Henle's loop, medullary and cortical thick ascending limbs of Henle's loop, and cortical, outer, and inner medullary collecting ducts; however, it was rarely detected in proximal convoluted tubules. Rbslo1 is most abundant in inner medulla. Expressed in Xenopus oocytes, rbslo1 generates depolarization-activated, outwardly rectifying K+ currents. Rbslo1 expressed in Chinese hamster ovary cells could be activated by depolarization and Ca2+. These data suggest that rbslo transcripts are expressed in multiple nephron segments and that the magnitude of mRNA expression varies among different nephron segments.
Asunto(s)
Clonación Molecular , Riñón/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Ratones , Datos de Secuencia Molecular , Oocitos , ARN Mensajero/metabolismo , Conejos , Distribución Tisular , XenopusRESUMEN
Glucagon-like peptide-1 (7-36) amide (GLP-1), in addition to its well known effect of enhancing glucose-mediated insulin release, has been shown to have insulinomimetic effects and to enhance insulin-mediated glucose uptake and lipid synthesis in 3T3-L1 adipocytes. To elucidate the mechanisms of GLP-1 action in these cells, we studied the signal transduction and peptide specificity of the GLP-1 response. In 3T3-L1 adipocytes, GLP-1 caused a decrease in intracellular cAMP levels which is the opposite to the response observed in pancreatic beta cells in response to the same peptide. In 3T3-L1 adipocytes, free intracellular calcium was not modified by GLP-1. Peptide specificity was examined to help determine if a different GLP receptor isoform was expressed in 3T3-L1 adipocytes vs. beta cells. Peptides with partial homology to GLP-1 such as GLP-2, GLP-1 (1-36), and glucagon all lowered cAMP levels in 3T3-L1 adipocytes. In addition, an antagonist of pancreatic GLP-1 receptor, exendin-4 (9-39), acted as an agonist to decrease cAMP levels in 3T3-L1 adipocytes as did exendin-4 (1-39), a known agonist for the pancreatic GLP-1 receptor. Binding studies using 125I-GLP-1 also suggest that pancreatic GLP-1 receptor isoform is not responsible for the effect of GLP-1 and related peptides in 3T3-L1 adipocytes. Based on these results, we propose that the major form of the GLP receptor in 3T3-L1 adipocytes is functionally different from the pancreatic GLP-1 receptor.
Asunto(s)
Adipocitos/metabolismo , Fragmentos de Péptidos , Péptidos/farmacología , Receptores de Glucagón/metabolismo , Transducción de Señal , Ponzoñas , Células 3T3 , Animales , Unión Competitiva , Células CHO , Calcio/metabolismo , Cricetinae , AMP Cíclico/metabolismo , Exenatida , Polipéptido Inhibidor Gástrico/farmacología , Glucagón/farmacología , Péptido 1 Similar al Glucagón , Receptor del Péptido 1 Similar al Glucagón , Péptidos Similares al Glucagón , Lipólisis/efectos de los fármacos , Ratones , Péptidos/metabolismo , Receptores de la Hormona Gastrointestinal/metabolismoRESUMEN
GLP-1-(7-36)-amide and exendin-4-(1-39) are glucagon-like peptide-1 (GLP-1) receptor agonists, whereas exendin-(9-39) is the only known antagonist. To analyze the transition from agonist to antagonist and to identify the amino acid residues involved in ligand activation of the GLP-1 receptor, we used exendin analogs with successive N-terminal truncations. Chinese hamster ovary cells stably transfected with the rat GLP-1 receptor were assayed for changes in intracellular cAMP caused by the test peptides in the absence or presence of half-maximal stimulatory doses of GLP-1. N-terminal truncation of a single amino acid reduced the agonist activity of the exendin peptide, whereas N-terminal truncation of 3-7 amino acids produced antagonists that were 4-10-fold more potent than exendin-(9-39). N-terminal truncation of GLP-1 by 2 amino acids resulted in weak agonist activity, but an 8-amino acid N-terminal truncation inactivated the peptide. Binding studies performed using 125I-labeled GLP-1 confirmed that all bioactive peptides specifically displaced tracer with high potency. In a set of exendin/GLP-1 chimeric peptides, substitution of GLP-1 sequences into exendin-(3-39) produced loss of antagonist activity with conversion to a weak agonist. The results show that receptor binding and activation occur in separate domains of exendin, but they are more closely coupled in GLP-1.
Asunto(s)
Hormonas Gastrointestinales/química , Fragmentos de Péptidos/química , Péptidos/química , Receptores de Glucagón/agonistas , Receptores de Glucagón/antagonistas & inhibidores , Ponzoñas/química , Secuencia de Aminoácidos , Animales , Unión Competitiva , Células CHO , Cricetinae , AMP Cíclico/metabolismo , Exenatida , Glucagón , Péptido 1 Similar al Glucagón , Receptor del Péptido 1 Similar al Glucagón , Péptidos Similares al Glucagón , Datos de Secuencia Molecular , Ratas , Proteínas Recombinantes de Fusión/química , Eliminación de Secuencia , Relación Estructura-Actividad , TransfecciónRESUMEN
Wistar rats develop glucose intolerance and have a diminished insulin response to glucose with age. The aim of this study was to investigate if these changes were reversible with glucagon-like peptide-1 (GLP-1), a peptide that we have previously shown could increase insulin mRNA and total insulin content in insulinoma cells. We infused 1.5 pmol/ kg-1.min-1 GLP-1 subcutaneously using ALZET microosmotic pumps into 22-mo-old Wistar rats for 48 h. Rat infused with either GLP-1 or saline were then subjected to an intraperitoneal glucose (1 g/kg body weight) tolerance test, 2 h after removing the pump. 15 min after the intraperitoneal glucose, GLP-1-treated animals had lower plasma glucose levels (9.04+/-0.92 mmol/liter, P < 0.01) than saline-treated animals (11.61+/-0.23 mmol/liter). At 30 min the plasma glucose was still lower in the GLP-1-treated animals (8.61+/-0.39 mmol/liter, P < 0.05) than saline-treated animals (10.36+/-0.43 mmol/liter). This decrease in glucose levels was reflected in the higher insulin levels attained in the GLP-1-treated animals (936+/-163 pmol/liter vs. 395+/-51 pmol/liter, GLP-1 vs. saline, respectively, P < 0.01), detected 15 min after glucose injection. GLP-1 treatment also increased pancreatic insulin, GLUT2, and glucokinase mRNA in the old rats. The effects of GLP-1 were abolished by simultaneous infusion of exendin [9-39], a specific antagonist of GLP-1. GLP-1 is therefore able to reverse some of the known defects that arise in the beta cell of the pancreas of Wistar rats, not only by increasing insulin secretion but also by inducing significant changes at the molecular level.
Asunto(s)
Envejecimiento/fisiología , Glucemia/metabolismo , Prueba de Tolerancia a la Glucosa , Péptidos/farmacología , Animales , Expresión Génica/efectos de los fármacos , Péptido 1 Similar al Glucagón , Glucoquinasa/genética , Glucosa/administración & dosificación , Transportador de Glucosa de Tipo 2 , Insulina/sangre , Insulina/genética , Insulina/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Péptidos/sangre , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
Glucagon-like peptide-1 (GLP-1), secreted from intestine in response to food intake, enhances insulin secretion from pancreatic beta-cells. In this study, we evaluated the effects of stably transfecting the GLP-1 receptor into an insulinoma cell line, RIN 1046-38, on basal and glucose-mediated insulin secretion and on second messenger pathways involved in insulin secretion. The GLP-1 receptor transfected cells had similar insulin mRNA levels but higher insulin content compared with parental cells. In GLP-1 receptor transfected cells, glucose (0.5 mM)-mediated insulin release was increased compared with parental cells (4.52 +/- 0.79 pmol insulin/l per mg protein x h vs. 2.21 +/- 0.36 pmol insulin/l per mg protein x h; mean +/- S.E., n = 6, P = 0.015, in transfected vs. parental cells, respectively). By hemolytic plaque assay measuring single cell insulin secretion, we observed that in the GLP-1 receptor transfected cells versus parental cells the increased insulin secretion was due to the presence of more glucose-responsive cells as well as more insulin released in response to glucose per cell. Resting intracellular cAMP was higher in the GLP-1 transfected cells (35.96 +/- 3.88 vs. 18.6 +/- 2.01 nmol/l per mg protein x h; mean +/- S.E., n = 4, P = 0.039, in transfected vs. parental cells, respectively). In response to GLP-1, both GLP-1 receptor transfected cells and parental cells showed increased cAMP levels independent of glucose. Resting intracellular calcium was the same in both parental and GLP-1 receptor transfected cells. However, more cells were responsive to glucose in the GLP-1 receptor transfected cells and the calcium transients attained in the presence of glucose developed at a faster rate and reached a higher amplitude than in parental cells. We conclude that having an excess of GLP-1 receptors renders beta-cells more sensitive to glucose.
Asunto(s)
Glucosa/farmacología , Insulina/metabolismo , Receptores de Glucagón/genética , Animales , Calcio/metabolismo , AMP Cíclico/metabolismo , Expresión Génica , Péptido 1 Similar al Glucagón , Receptor del Péptido 1 Similar al Glucagón , Secreción de Insulina , Insulinoma/genética , Insulinoma/metabolismo , Transporte Iónico/efectos de los fármacos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Péptidos/farmacología , Ratas , Transfección , Células Tumorales CultivadasRESUMEN
In this study, a partial hamster complementary DNA encoding ERCC-1, a member of the DNA excision repair gene family, has been cloned. The nucleic acid and amino acid sequences were highly homologous to those of human and mouse ERCC-1. The hamster ERCC-1 gene was expressed as a 1.2-kilobase message in cultured Chinese hamster ovary cells. Northern (RNA) blot analysis revealed that overexpression of the insulin receptor or various growth factor receptor tyrosine kinases in Chinese hamster ovary cells increased ERCC-1 messenger RNA (mRNA) levels. This effect did not occur in cells overexpressing mutated insulin receptors that are known to have impaired kinase-related signaling. Increased ERCC-1 expression correlated with resistance to UV exposure. Fluorescent-activated cell sorter analysis of confluent cell populations indicated no differences in cell cycle distribution. Furthermore, no significant relationship was demonstrated between the relative expression of ERCC-1 mRNA and the rate of glucose utilization. Insulin enhanced the accumulation of ERCC-1 mRNA in serum-deprived cells expressing wild-type insulin receptors. The potential role for activation of the insulin receptor and related growth factor receptors in ERCC-1 gene expression and function remains to be defined.
Asunto(s)
Proteínas de Unión al ADN , Endonucleasas , Expresión Génica , Proteínas/genética , ARN Mensajero/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Células CHO , Cricetinae , Reparación del ADN , ADN Complementario/química , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Humanos , Insulina/farmacología , Ratones , Datos de Secuencia Molecular , Mutación , Proteínas/química , Rayos UltravioletaRESUMEN
HT29 cells endogenously express the cystic fibrosis transmembrane conductance regulator (CFTR) and have been used previously as a model to examine cellular regulation of CFTR expression and chloride secretory function. Homologous recombination has been used to specifically disrupt CFTR transcription in the HT29-18-C1 subclone. Experiments demonstrate successful disruption of a CFTR allele by DNA constructs, which target insertion of the neomycin phosphotransferase gene into CFTR exon 1 via homologous recombination. The mutation of one allele is a partial knockout because this cell line has multiple CFTR alleles. The mutation is confirmed by polymerase chain reaction (PCR) and genomic Southern blot analysis. A 52-68% reduction in CFTR mRNA levels is observed in the mutant cell line by both Northern and PCR analysis. However, Western blots show no decrease in total CFTR protein levels. Consistent with the lack of reduction in CFTR protein, the partial knockout mutant does not demonstrate alterations in cyclic AMP or calcium stimulation of chloride efflux or net osmolyte loss. Results suggest that posttranscriptional regulation of CFTR levels may contribute to maintenance of cellular chloride transport function.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Vectores Genéticos , Células HT29/fisiología , Alelos , Empalme Alternativo/genética , Northern Blotting , Western Blotting , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Células Epiteliales , Regulación de la Expresión Génica/genética , Pruebas Genéticas , Humanos , Mutagénesis/fisiología , Fenotipo , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Recombinación Genética , TransfecciónRESUMEN
Glucagon-like peptide-1 (7-36 amide) (GLP-1) is known to increase insulin release when given as a bolus in the fasted and fed state. GLP-1 also increases glucose uptake and lipid synthesis in cultured adipocytes. In this study we investigated the effects of GLP-1 on glucose uptake and on the levels of expression of the facilitative glucose transporters, GLUT1 and GLUT4, in fully differentiated 3T3-L1 adipocytes. Cells were incubated with GLP-1 (10 nM) with or without insulin (10 and 100 nM) for 24 h. Under these conditions, GLP-1 alone caused an increase in basal and acute insulin-stimulated glucose uptake along with an increase in GLUT1 and GLUT4 protein levels. However, there was no change in the expression of GLUT1 and GLUT4 mRNAs. In the absence of GLP-1, prolonged exposure to insulin caused a marked reduction in the levels of GLUT4 mRNA and protein, and an inhibition of glucose uptake after an acute exposure to insulin. This insulin-induced down-regulation of GLUT4 was prevented when GLP-1 was present during the 24-h treatment. In contrast, the acute insulin-stimulated glucose uptake could not be restored by GLP-1. GLP-1 is therefore the first gut hormone shown to be capable of modulating glucose transporter levels in cultured adipocytes.
Asunto(s)
Adipocitos/citología , Adipocitos/metabolismo , Hexosas/farmacocinética , Proteínas de Transporte de Monosacáridos/genética , Proteínas Musculares , Células 3T3 , Animales , AMP Cíclico/metabolismo , Desoxiglucosa/farmacocinética , Regulación de la Expresión Génica , Glucagón/farmacología , Péptido 1 Similar al Glucagón , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Hipoglucemiantes/farmacología , Insulina/metabolismo , Insulina/farmacología , Ratones , Proteínas de Transporte de Monosacáridos/efectos de los fármacos , Proteínas de Transporte de Monosacáridos/metabolismo , Fragmentos de Péptidos/farmacología , Unión Proteica , Precursores de Proteínas/farmacología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Receptor de Insulina/genética , Receptor de Insulina/metabolismoRESUMEN
The role of the insulin receptor COOH-terminal domain in the regulation of insulin signal transduction was explored with a variety of synthetic peptides. One of the peptides, termed peptide HC, whose structure corresponds to residues 1293-1307 of the insulin proreceptor sequence, enhanced insulin-stimulated autophosphorylation of the insulin receptor in cell-free systems and in semipermeabilized Chinese hamster ovary (CHO) cells that had been transfected with an expression plasmid encoding the human insulin receptor (CHO/HIRc) at concentrations where there was no detectable effect on basal autophosphorylation levels or on receptor dephosphorylation. A lipophilic analogue of peptide HC, stearyl peptide HC, added to intact CHO/HIRc cells enhanced significantly insulin-stimulated insulin receptor autophosphorylation while having no effect on ligand-stimulated receptor phosphorylation in CHO cells overexpressing either the IGF-1 receptor or epidermal growth factor receptor. Addition of stearyl peptide HC to CHO/HIRc cells resulted in a 2.4 +/- 0.3-fold increase in the amount of insulin-stimulated phosphatidylinositol 3-kinase detected in anti-IRS-1 immunoprecipitates and a 2.1 +/- 0.6-fold increase in the levels of tyrosine phosphorylation of mitogen-activated protein kinase in response to insulin. Finally, a derivative of peptide HC coupled to a biotin moiety was prepared and showed to bind with the beta-subunit of the wild-type insulin receptor and a truncated receptor that lacks 43 amino acids from its carboxyl terminus. However, there was little binding, if any, of the peptide with the IGF-1 receptors or the epidermal growth factor receptors. Taken together, our data demonstrate that a pentadecapeptide related to the carboxyl terminus of the insulin receptor binds to the insulin receptor beta-subunit and that this interaction may contribute to the increased receptor's intrinsic activity and signal transduction.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos , Fragmentos de Péptidos/metabolismo , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Transducción de Señal , Animales , Biotina , Células CHO , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Cricetinae , Humanos , Insulina/metabolismo , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Ácidos Esteáricos , Tirosina/metabolismoRESUMEN
Primary culture of rat islets of Langerhans lose glucose responsiveness and eventually die when cultured for a long period of time. In this study we evaluated the effect of matrigel, a basement membrane extract, on (i) islet cell survival, (ii) cell responsiveness following a glucose challenge, and (iii) mRNA levels for insulin, glucagon, and somatostatin. Pancreatic islets were isolated by collagenase digestion and plated in culture dishes either coated or not with a matrigel layer. Using the reverse hemolytic plaque assay, we determined the total number of insulin-secreting cells and the amount of insulin secreted by individual beta cells. After 1 h of exposure to 5 mM glucose, beta cells from 6-month-old rat islets cultured for 6 weeks on matrigel showed an equal number of insulin-secreting cells compared to freshly isolated islets cultured for only 3 days in the absence of matrigel (39.5 +/- 2.5 vs. 37.1 +/- 2.6%). Furthermore, the release of insulin by cells cultured on matrigel for 6 weeks increased in a glucose-dependent manner (p < 0.001) and showed an ED50 of 7 mM. However, the amount of insulin released per single beta cell was reduced by 40-60% (p < 0.02) compared to that released from isolated beta cells derived from a 3-day culture of islets. Finally, there was a 35-55% increase (p < 0.05) in the levels of insulin, glucagon, and somatostatin mRNAs in cells cultured for 6 weeks on matrigel. These data suggest a trophic effect of matrigel on the maintenance of normal beta-cell activity and function and may lead the way to the development of a new model for the study of pancreatic islets in long-term culture.
Asunto(s)
Insulina/genética , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Animales , Secuencia de Bases , Colágeno , Técnicas de Cultivo/métodos , ADN Complementario/genética , Combinación de Medicamentos , Matriz Extracelular , Glucagón/genética , Secreción de Insulina , Laminina , Masculino , Proteoglicanos , Ratas , Ratas Wistar , Somatostatina/genéticaRESUMEN
Acute studies of glucose-dependent insulinotropic peptide (GIP) have shown that GIP can synergize with glucose in stimulating insulin secretion both in vivo and in vitro. Here we studied the effects of extended exposure of RIN 1046-38 cells, an insulin-secreting cell line, to GIP and the mechanisms by which GIP synergizes with glucose in stimulating insulin secretion. Incubation of the cells with 100 nM GIP in the presence of glucose for 12 h significantly increased insulin release (287 +/- 31.7 vs. 102 +/- 9.7 ng/mg protein; n = 3), intracellular insulin content (12.8 +/- 0.83 vs. 8.2 +/- 0.52 ng/mg protein; n = 3), and insulin mRNA (approximately 2.7-fold; 24 h incubation) when compared to cells cultured with glucose alone. The insulinotropic effects of GIP on RIN 1046-38 cells were accompanied by an up-regulation of GLUT-1 and hexokinase I mRNA (1.75-fold) compared to non-GIP-treated cells; mRNA levels of GLUT-2 and glucokinase were unchanged by GIP, in the presence or absence of glucose. Our study suggests that the mechanism by which extended exposure of RIN 1046-38 cells to GIP increases glucose-stimulated insulin secretion includes up-regulation of glucose sensing elements.